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2021.03.12.435067.Full.Pdf bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435067; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 A Fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the 2 L-arginine biosynthetic pathway of Mycobacterium tuberculosis 3 4 Pooja Gupta1, Sherine E. Thomas1, James Cory-Wright1, Víctor Sebastián-Pérez1,2, Ailidh Burgess1, 5 Emma Cattermole1, Clio Meghir1, Chris Abell3, Anthony G. Coyne3, William R. Jacobs Jr. 4 Tom L. 6 Blundell1, Sangeeta Tiwari5* and Vítor Mendes1#*. 7 8 1 Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, 9 UK 10 2 Centro de Investigaciones Biológicas Margarita Salas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, 11 Spain. 12 2 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, 13 USA 14 3 Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 15 1EW, UK 16 4 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, 17 USA. 18 5 Department of Biological Sciences & Border Biomedical Research Centre, University of Texas at El 19 Paso, El Paso, TX 79968, USA. 20 # Current affiliation: MRC-Laboratory of Molecular Biology, Molecular Immunity Unit, Francis Crick 21 Ave, Cambridge, CB2 0QH, UK. 22 23 * To whom correspondence should be addressed 24 Vitor Mendes: [email protected]; +44 1223267723 25 Sangeeta Tiwari: [email protected]; +1 9157476889 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435067; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 27 Abstract 28 The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L- 29 glutamate to L-arginine, appears to be attractive target for anti-Tuberculosis (TB) drug discovery. 30 Starvation of M. tuberculosis deleted for either argB or argF genes led to rapid sterilization of these 31 strains in mice while Chemical inhibition of ArgJ with Pranlukast was also found to clear chronic M. 32 tuberculosis infection in animal models. In this work, the ligandability of four enzymes of the pathway 33 ArgB, ArgC, ArgD and ArgF is explored using a fragment-based approach. We reveal several hits for 34 these enzymes validated with biochemical and biophysical assays, and X-ray crystallographic data, 35 which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. 36 These results demonstrate the potential of more enzymes in this pathway to be targeted with 37 dedicated drug discovery programmes. 38 39 Keywords 40 ArgB, ArgC, ArgD, ArgF, Mycobacterium tuberculosis, FBDD 41 42 Abbreviations 43 TB – tuberculosis; FBDD – Fragment-based drug discovery; DSF – Differential scanning fluorimetry; 44 ASU - asymmetric unit; SPR – Surface plasmon resonance; NMR – Nuclear magnetic resonance; ITC – 45 Isothermal titration calorimetry. 46 47 48 49 50 51 52 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435067; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 53 1. Introduction 54 Despite the availability of effective chemotherapy, tuberculosis (TB) remains a leading infectious cause 55 of morbidity and mortality worldwide. In 2019, an estimated 1.2 million deaths were caused by TB, 56 and an additional 208,000 were a result of HIV-TB co-infection (1). Simultaneously, the existing 57 multidrug treatment regimen has a success rate of 85% in drug-sensitive TB cases (in the 2018 cohort), 58 drug toxicity, a long treatment duration, and resulting patient non-compliance, as well as 59 incompatibility with antiretroviral therapy all compromise its effectiveness. Alarmingly, the 60 emergence of multi-drug resistant (MDR) and extensively-drug-resistant (XDR) strains of 61 Mycobacterium tuberculosis has further undermined the efficacy of current antitubercular therapy: 62 only 57% of MDR cases were successfully treated worldwide in the 2017 cohort. New antitubercular 63 agents are therefore urgently required and novel chemical scaffolds and mechanisms of action must 64 be identified that can shorten therapy and circumvent development of drug resistance. While many 65 drugs can be bacteriocidal, M. tuberculosis has the ability to generate subpopulations that enter into 66 a persister state making them phenotypically drug resistant (2). The consideration of preventing 67 persister formation or killing persisters needs to be addressed in future drug discovery campaigns 68 against M .tuberculosis. 69 M. tuberculosis, like the leprosy bacillus, has retained its ability to make all 20 amino acids and most 70 vitamins. This retention of these biosynthetic genes reflect a evolutionary pressure suggesting the 71 pathogenic mycobacteria have chosen not to obtain amino acids or many vitamins from the host and 72 has thus been described as an autarkic lifestyle (3). However, not all amino acid auxotrophies behave 73 the same. Several amino acid auxotrophs were found to have attenuated virulence inside host 74 organisms, suggesting that while enzymes in amino acid biosynthetic pathways are essential in vitro, 75 the pathogen can scavenge amino acids (albeit insufficiently) inside the host and survive (4-9). 76 However, it was shown that methionine and arginine auxotrophs of M. tuberculosis are rapidly 77 sterilised in both immunocompetent and immunodeficient (SCID) mice without the appearance of 78 suppressor/bypass mutants (3, 10). Despite the presence of two arginine transporters in M. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435067; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 79 tuberculosis (11, 12) and sufficiently high serum concentrations of arginine in the host (13), the 80 virulence of ΔargB or ΔargF mutants is entirely abolished as arginine deprivation results in extensive 81 oxidative damage (10). The case for drug discovery approaches to target arginine biosynthetic 82 enzymes is further bolstered by work demonstrating that chemical inhibition of ArgJ with Pranlukast, 83 a cysteinyl leukotriene receptor-1 antagonist use to treat asthmatic exacerbations, cleared a chronic 84 M. tuberculosis infection in BALB/c mice (14). The arginine biosynthesis pathway consists of eight 85 different enzymes (Figure 1A) all considered to be essential for M. tuberculosis growth in vitro (15). 86 Except argA which encodes the first enzyme of the pathway, all other genes are present in a single 87 operon that also includes the repressor argR (Figure 1B). 88 Fragment based drug discovery (FBDD) is now an established lead-generation strategy in both industry 89 and academia, having yielded over 30 compounds in clinical trials, including approved cancer drugs 90 like vemurafenib, Kisqali, Balversa and venetoclax (16). This approach consists of screening a library 91 of small molecules (150-300 Da) against a target of interest using biophysical, biochemical and 92 structural biology methods. The low complexity of fragments allows for efficient exploration of the 93 chemical space of the target, often revealing unexpected binding sites in proteins. Although fragments 94 often bind weakly, they tend to bind to hotspot regions of the target, forming well defined interactions 95 that allow subsequent elaboration into larger, drug-like molecules (17, 18). Our group and a few others 96 have pioneered using this approach against different mycobacterial species and different protein 97 targets with varying degrees of success (18-26). 98 Using this approach, we have screened four enzymes of the arginine biosynthesis pathway not yet 99 explored drug discovery programmes: ArgB, ArgC, ArgD and ArgF. Herein we report the structures of 100 the four enzymes in complex with fragments hits, including a novel allosteric site of ArgB and allosteric 101 inhibitors of this enzyme. Importantly, this work also assesses the potential of these enzymes as 102 candidates of future drug discovery programmes. 103 104 2. Materials and Methods 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435067; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 105 2.1. Molecular Cloning 106 The argB gene was amplified from chromosomal DNA of M. tuberculosis H37Rv strain obtained from 107 ATCC (ATCC25618D-2) while the ORFs of argC, argD and argF were purchased as E. coli codon- 108 optimised synthetic gene strings through the ThermoFisher GeneArt Gene Synthesis service. The argB 109 gene was cloned into pHAT4 (27) using NcoI and XhoI sites. The gene strings of argC, argD and argF 110 were cloned into a pET28a vector (modified to include an N-terminal 6xhis SUMO) (28) using BamHI 111 and HindIII restriction sites. All constructs were confirmed by sequencing. 112 113 2.2. Protein expression and purification 114 250 mL of autoclaved 2xYT broth (Formedium) prepared in distilled water, containing 100 μg/mL 115 ampicillin for pHAT4:argB or 30 μg/mL kanamycin for pET28a:argC/argD/argF, was inoculated with E.
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